PROJECT SUMMARY Tissue acidosis is a major contributing factor to neuronal cell death associated with neurological diseases, such as stroke, traumatic brain and spinal cord injuries, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), as well as Alzheimer's, Huntington, and Parkinson?s diseases. It has been well established that acid-sensing ion channels subtype 1a (ASIC1a) is critically involved in acidosis-induced neuronal cell death in both in vitro and in vivo models. The protective effects of ASIC1a knockout and pharmacological inhibition of ASIC1a function shown in the mouse models of ischemic stroke, MS, HD, and ALS testify the potential of targeting ASIC1a to mitigate neuronal damages in multiple types of neurological disorders. However, the mechanism(s) by which ASIC1a activation causes neuronal death remains mysterious despite extensive investigations. Conventionally, ASIC1a is believed to form cell surface cation channels activated by extracellular protons to mediate Na+ and Ca2+ entry into the cell. The ion conducting function, especially Ca2+ influx, is thought to cause Ca2+ overload that eventually leads to acid-induced cytotoxicity. However, our recent results suggest that the cell killing effect of ASIC1a is dependent not on its channel conductance, but on the recruitment and phosphorylation of serine/threonine kinase receptor interaction protein 1 (RIP1) to the C-terminus of ASIC1a protein. RIP1 is a key mediator of death receptor-induced necroptotic pathway. In rodent model of ischemic stroke, middle cerebral artery occlusion (MCAO), inhibiting RIP1, just like inhibiting ASIC1a, was shown to be neuroprotective even when the drug was administered several hours after the onset of brain ischemia. Therefore, acidosis neuronal death most likely occurs through ASIC1a-RIP1 physical coupling and the consequent activation of RIP1-dependent necroptosis. The goal of the proposed project is to elucidate this novel mechanism of acid-induced, ASIC1a/RIP1-mediated necroptotic cell demise in neurons. Aim I will define the death pathway mediated by ASIC1a-RIP1 interaction in response to acidosis through systematic evaluation of key factors involved in ASIC1a-mediated cell demise in cultured neurons and in the mouse MCAO model. Aim II will examine a novel mechanism by which a chaperone protein facilitates RIP1 activation through disruption of an intramolecular interaction between the cytoplasmic N- and C-termini of ASIC1a. Aim III will elucidate how the C-terminal RIP1 interaction domain of ASIC1a triggers and mediates acidosis-induced necroptosis and test whether disrupting such interaction can mitigate neuronal damage caused by acidosis and brain ischemia. Successful completion of this project will provide a better understanding of the molecular mechanism of neuronal acidotoxicity, which will shed lights on new treatment strategies for several major types of neurological disorders.